FORMULATION AND EVALUATION OF CHITOSAN NANOPATICLES OF A BROAD SPECTRUM ANTIBACTERIAL

DEVELOPMENT AND EVALUATION OF NANOPARTICULATE DRUG DELIVERY SYSTEM CONTAINING AN ANTICANCER DRUG

SYNOPSIS FOR

M. PHARM. DISSERTATION

SUBMITTED TO

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

KARNATAKA

BY

AMBIKA T.V.

I M. PHARM.

Department of Pharmaceutics

Dayananda Sagar College of PharmacY

2009

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BANGALORE, KARNATAKA

ANNEXURE-II

PROFAMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1.

Name of the candidate and address (in block letters)

AMBIKA T.V.,

I M. PHARM.,

DEPARTMENT OF PHARMACEUTICS,

DAYANANDA SAGAR COLLEGE OF PHARMACY,

KUMARASWAMY LAYOUT,

BANGALORE-560078.

PERMANENT ADDRESS

# 96, ARUNODAYA NILAYA,

kRISHNAPPA LAYOUT,

TINDLU, VIDYARANYAPURA POST,

BANGALORE-560097

2.

Name of the institute

Dayananda Sagar College of Pharmacy, Shavige Malleswara Hills,

Kumaraswamy Layout,

Bangalore-560078,

Karnataka.

3.

Course of study and subject

Master of Pharmacy in Pharmaceutics

4.

Date of admission to course

23rd May 2009

5.

Title of the project:

“DEVELOPMENT AND EVALUATION OF NANOPARTICULATE DRUG DELIVERY SYSTEM CONTAINING AN ANTICANCER DRUG”

6.

Brief resume of the intended work:

6.1 Need of the study:

Cancer is a generic term for a large group of diseases that can affect any part of the body. Cancer is responsible for about 25% of all deaths in the U.S., and is a major public health problem in many parts of the world. Cancer is a leading cause of death worldwide. More than 70% of all cancer deaths occurred in low- and middle-income countries. Deaths from cancer worldwide are projected to continue rising, with an estimated 12 million deaths in 2030.

Treatment of cancer includes chemotherapy, radiation therapy, gene therapy, photodynamic therapy, biologic therapy, surgical removal of tumor cells, etc. Chemotherapy is the most convenient and non-expensive when compared to other modes of treatment. Varieties of anticancer drugs are available in the market and some of them are under clinical trials. The main problem with anti-cancer drugs is that they not only affect the cancerous cells but also affect the normal cells. These happen due to non-specific targeting to cancerous cells and hence other normal cells get affected.

Recently, drug targeting especially targeting of drugs by nanoparticles have been getting much attention by the researchers for treating cancer. A critical advantage in treating cancer with nanoparticles is the inherent leaky vasculature present serving cancerous tissues. The defective vascular architecture, created due to rapid vascularization necessary to serve fast-growing cancers, coupled with poor lymphatic drainage allows an enhanced permeation and retention effect.

Targeting the tumor vasculature is a strategy that can allow targeted delivery to a wide range of tumor types. Tremendous opportunities exist for using nanoparticles as controlled drug delivery systems for cancer treatment. Natural and synthetic polymers including albumin, fibrinogen, alginate, chitosan, and collagen have been used for the fabrication of nanoparticles.

Hence considering the importance of treating caner, an attempt will be made to target and deliver an anti-cancer drug to these cancerous cells so as to minimize adverse effects, dose and also dosing frequency. This is achieved by formulating anticancer drugs in the form of nanoparticles.

6.2 Review of literature

Adriamycin1 nanoparticles were prepared using biocompatible and biodegradable poly(lactide-co-glycolide)-polyethylene glycol (PLGA-PEG) polymers. The nanoparticles were prepared by precipitation-solvent evaporation technique. Particle sizes were between 65 and 100 nm for different compositions of PLGA-PEG copolymers. Entrapment efficiency was 25%–33%. Adriamycin release from the nanoparticles at pH 7.4 showed an initial burst release and then sustained release phase. These results showed that PLGA-PEG nanoparticles could be an effective carrier for cancer therapy.

Busulfan2 nanoparticles were formulated by precipitating into five different types of poly(alkyl cyanoacrylate) polymers. Poly(isobutyl cyanoacrylate) (PIBCA) and poly(ethyl cyanoacrylate) polymers showed good loading efficiency. Molecular modeling along with energy minimization process was employed to identify the nature of the interactions occurring between busulfan and PIBCA. Optimization studies showed PIBCA nanoparticles displaying busulfan loading ratios equal to 5.9% (w/w) with percentage yield to be 71% (w/w). H-NMR spectroscopy was done to show the chemical integrity of the drug was preserved after loading into nanoparticles. The in vitro release studies under sink conditions, in water, or in rat plasma showed a fast release in the first 10 minutes followed by as slower one over 6 hours.

Carmustine3-loaded poly(lactic acid) (PLA) nanoparticles were formulated by spontaneous emulsification-solvent diffusion method using Pluroinc F68 as emulsifying agent. Micellar behavior of surfactant in aqueous solution on the sizes of nanoparticles was studied. Results revealed that F 68 dissolved in water or sodium acetate-acetic media with pH of 5.0 to 5.5 resulted in larger PLA particles (226nm) or clumps. In acidic media with ph 4.0 and 4.5 ultrasmall particles with size 50 and 80 nm was obtained. The change in size had less effect on drug content and entrapment efficiency, and showed strong enhanced cytotoxic effect of the loaded drug in vitro, and the effect being more relevant for prolonged incubation times.

Lipid nanoparticles of the anticancer drug Chlorambucil 4(CLB) were prepared by ultrasonication, using stearic acid as the core lipid. Stearic acid solid nanoparticles (SLN), sterically stabilized SLN with pegylated phospholipids as stabilizer, nanostructured lipid complexes with oleic acid as adjunct lipid, lipid nanocomplexes with dimethyl dioctadecyl ammonium bromide (DDAB) as surface modifier (LN). Lipid nanoparticles were characterized for particle size, assay and encapsulation efficiency, particle morphology and physico-chemical stability over 90 days. All formulations were physically stable, with an average particle size of 147 (±10) nm. The drug encapsulation efficiency (DEE) of all formulations except LN decreased significantly probably due to the expulsion of CLB upon crystallization. This indicated that the presence of DDAB in stearic acid nanoparticles increases DEE, preventing CLB degradation in the aqueous disperse phase. Pharmacokinetic studies of the intravenous LN formulation revealed plasma clearance kinetics were comparable to that of CLB solution, indicating electrostatic charge mediated clearance, as reported earlier. In tissue and tumor distribution studies, lower AUC values of CLB were observed for LN compared to CLB solution in liver, kidneys, heart and lungs. Higher AUC values of LN formulation as compared to CLB solution in tumors suggested that the presence of DDAB on the lipid nanoparticles resulted in greater accumulation of the drug in tumors.

The in vitro anticancer activity of cisplatin5-loaded PLGA-mPEG nanoparticles on human prostate cancer LNCaP cells was investigated. The uptake of the PLGA-mPEG nanoparticles by the LNCaP cells was also studied. Blank PLGA-mPEG nanoparticles exhibited low cytotoxicity, which increased with increasing PLGA/PEG ratio in the PLGA-mPEG copolymer used to prepare the nanoparticles. PLGA-mPEG nanoparticles loaded with cisplatin exerted in vitro anticancer activity against LNCaP cells that was comparable to the activity of free (non-entrapped in nanoparticles) cisplatin. Little differences in the in vitro anticancer activity of the different nanoparticle compositions were found. Visual evidence of nanoparticles’ uptake by the LNCaP cells was obtained with nanoparticles labeled with PLGA(4165)-PyrBu(274) or dextran-rhodamine B isothiocyanate using fluorescence microscopy.

An attempt was made to evaluate the potential of chitosan nanoparticles as carriers for the anthracycline drug, doxorubicin6 (DOX). They entrapped a cationic, hydrophilic molecule into nanoparticles formed by ionic gelation of the positively charged polysaccharide chitosan. Hence attempt was made to mask the positive charge of DOX by complexing it with the polyanion, dextran sulfate. This modification doubled DOX encapsulation efficiency relative to controls and enabled real loadings up to 4.0 wt % DOX. Separately chitosan and DOX was investigated for any complexes prior to the formation of the particles. No dissociation of the complex was observed upon formation of the nanoparticles. Fluorimetric analysis of the drug released in vitro showed an initial release phase, followed by a very slow release. The evaluation of the activity of DOX-loaded nanoparticles in cell cultures indicated that those containing dextran sulfate were able to maintain cytostatic activity relative to free DOX, while DOX complexed to chitosan before nanoparticle formation showed slightly decreased activity. Additionally, confocal studies showed that DOX was not released in the cell culture medium but entered the cells while remaining associated to the nanoparticles. The preliminary studies showed the feasibility of chitosan nanoparticles to entrap the basic drug DOX and to deliver it into the cells in its active form.

Methotrexate7 (MTX)-encapsulated polymeric nanoparticles using methoxy poly(ethylene glycol) (MPEG)-grafted chitosan (ChitoPEG) copolymer. MTX-encapsulated polymeric nanoparticles of ChitoPEG copolymer has around 50–300 nm particle size and showed spherical shape when observed by transmission electron microscope. Nuclear magnetic resonance study indicated that MTX was complexed with chitosan and core–shell type nanoparticles was formed in aqueous environment, i.e., MTX/chitosan complexes composed of inner-core and MPEG composed of outer-shell of the nanoparticles. Loading efficiency of MTX in the polymeric nanoparticles was 94% (w/w) of ChitoPEG-1, 91.1% (w/w) of ChitoPEG-2, 90.1% (w/w) of ChitoPEG-3 and 65.2% (w/w) of ChitoPEG-4. Higher the drug feeding ratio, higher is the drug content and lower the loading efficiency. Higher the MPEG graft ratio in the copolymer, lower the drug content and loading efficiency. Drug contents evaluated by NMR were same as found by UV spectrophotometer.

Chitosan nanoparticles containing the anticancer drug paclitaxel8 were prepared by solvent evaporation and emulsification cross-linking method. Uniform nanoparticles with an average particle size of 116 ± 15 nm with high encapsulation efficiencies (EE) were obtained. A sustained release of paclitaxel from paclitaxel-loaded chitosan nanoparticles was successful. Using different ratios of paclitaxel-to-chitosan, the EE ranged from 32.2 ± 8.21% to 94.0 ± 16.73 %. The drug release rates of paclitaxel from the nanoparticles were approximately 26.55 ± 2.11% and 93.44 ± 10.96% after 1 day and 13 days respectively, suggesting sustained drug delivery system. Cytotoxicity tests showed that the paclitaxel-loaded chitosan had higher cell toxicity than the individual paclitaxel and confocal microscopy analysis confirmed excellent cellular uptake efficiency. Flow cytometric analysis revealed two subdiploid peaks for the cells in the paclitaxel-loaded nanoparticles and paclitaxel treated groups, respectively, with the intensity of the former higher than that of the latter. Moreover, the cell cycle was arrested in the G2-M phase, which was consistent with the action mechanism of the direct administration of paclitaxel. These results indicate that chitosan nanoparticles have potential uses as anticancer drug carriers and also have an enhanced anticancer effect.

Mitomycin C9 (MMC) loaded PLA nanoparticles were prepared by a new single emulsion solvent evaporation method, in which soybean phosphatidylcholine (SPC) was employed to improve the liposolubility of MMC. Four main influential factors based on the results of a single-factor test, namely, PLA molecular weight, ratio of PLA to SPC (wt/wt) and MMC to SPC (wt/wt), volume ratio of oil phase to water phase, were evaluated using an orthogonal design with respect to drug entrapment efficiency. The drug release study was performed in pH 7.2 PBS at 37 °C with drug analysis using UV/vis spectrometer at 365 nm. MMC-PLA particles prepared by classical method were used as comparison. The formulated MMC-SPC-PLA nanoparticles under optimized condition are found to be relatively uniform in size (594 nm) with up to 94.8% of drug entrapment efficiency compared to 6.44 μm of PLA-MMC microparticles with 34.5% of drug entrapment efficiency. The release of MMC shows biphasic with an initial burst effect, followed by a cumulated drug release over 30 days is 50.17% for PLA-MMC-SPC nanoparticles, and 74.1% for PLA-MMC particles. The IR analysis of MMC-SPC complex shows that their high liposolubility may be attributed to some weak physical interaction between MMC and SPC during the formation of the complex. It is concluded that the new method is advantageous in terms of smaller size, lower size distribution, higher encapsulation yield, and longer sustained drug release in comparison to classical method.

Nano-sized poly (D, L lactide-co-glycolide) (PLGA) particles containing estrogen10 were prepared employing emulsification–diffusion method. Estrogen was chosen as a model drug. The preparation method consists of emulsifying a solution of polymer and drug in the aqueous phase containing stabilizer, previously saturated, followed by adding excess water. Influence of process variables on the mean particle size of nanoparticles were studied. It was clarified that the type and concentrations of stabilizer, homogenizer speed, and polymer concentration determined the size of PLGA nanoparticles. Especially when didodecyl dimethyl ammonium bromide (DMAB) was used as a stabilizer, estrogen containing nanoparticles of smaller than 100 nm was obtained.

A study described the preparation of antibacterially active emulsified polyacrylate nanoparticles in which a penicillin11 antibiotic is covalently conjugated onto the polymeric framework. These nanoparticles were prepared in water by emulsion polymerization of an acrylated penicillin analogue pre-dissolved in a 7:3 mixture of butyl acrylate and styrene in the presence of sodium dodecyl sulfate (surfactant) and potassium persulfate (radical initiator). Dynamic light scattering analysis and atomic force microscopy images show that the emulsions contain nanoparticles of approximately 40 nm in diameter. The nanoparticles have equipotent in vitro antibacterial properties against methicillin-susceptible and methicillin-resistant forms of Staphylococcus aureus and indefinite stability towards β-lactamase.

The estradiol12(E2)-loaded chitosan nanoparticles (CS-NPs) were prepared by ionic gelation of chitosan with tripolyphosphate anions (TPP). The CS-NPs had a mean size of (269.3 ± 31.6) nm, a zeta potential of +25.4 mV, and loading capacity of E2 CS-NPs suspension was 1.9 mg ml_1, and entrapment efficiency was 64.7% on average. They also investigated the levels of E2 in blood and the cerebrospinal fluid (CSF) in rats. The plasma levels achieved following intranasal administration (32.7 ± 10.1 ng ml_1; tmax 28 ± 4.5 min) were significantly lower than those after intravenous administration (151.4 ± 28.2 ng ml_1), while CSF concentrations achieved after intranasal administration (76.4 ± 14.0 ng ml_1; tmax 28 ± 17.9 min) were significantly higher than those after intravenous administration (29.5 ± 7.4 ng ml_1 tmax 60 min). The drug targeting index (DTI) of nasal route was 3.2, percent of drug targeting (DTP%) was 68.4%. These results showed that the E2 must be directly transported from the nasal cavity into the CSF in rats. Finally, compared with E2 inclusion complex, CS-NPs improved significantly E2 being transported into central nervous system (CNS).

The ammonium glycyrrhizinate13-loaded chitosan nanoparticles were prepared by ionic gelation of chitosan with tripolyphosphate anions. The particle size and zeta potential of nanoparticles were determined by dynamic light scattering and a zeta potential analyzer respectively. The effects including chitosan molecular weight, chitosan concentration, ammonium glycyrrhizinate concentration and polyethylene glycol (PEG) on the physicochemical properties of the nanoparticles were studied. These nanoparticles have ammonium glycyrrhizinate loading efficiency. The encapsulation efficiency decreased with the increase of ammonium glycyrrhizinate concentration and chitosan concentration. The introduction of PEG can decrease significantly the positive charge of particle surface. These studies showed that chitosan can complex TPP to form stable cationic nanoparticles for subsequent ammonium glycyrrhizinate loading.

6.3 Objective of the study:

The objective of the study is to develop nanoparticles for anticancer drug, which are expected to

· Maintain the therapeutic drug concentration in the blood for a prolonged period of time.

· Improve bioavailability.

· Improve the efficacy of the drug.

· Reduce the dose related side effects.

· Targeting the drug to the tumor.

6.4 Plan of work

The work will be executed as follows:

· Selection of suitable drug and polymer for the preparation of nanoparticles.

· Preformulation studies.

· Formation of Dummy nanoparticles.

· Formulation of different batches of nanoparticles of anticancer drug.

· Evaluation of formulated nanoparticles include:

· Process yield

· Particle size analysis

· Percentage of drug loading

· In-vitro drug release studies

· Release kinetics

7.

Materials and methods

7.1 Source of data:

Official Pharmacopoeia, Standard books, Pharmaceutical databases, internet, etc.

7.2 Method of collection of the data (including sampling procedure, if any):

The pharmacological details of the drug will be collected from various standard books, journals and other sources like research literature databases such as Medline, Science Direct, etc.

Experimental data will be collected from the evaluation of designed formulation and then subjecting the formulation to different studies such as preformulation, drug content, release profile, stability studies, particle size, etc.

The outline of such methods that would be adopted includes;

1. Preformulation studies standard to development of nanoparticles.

2. Selection of suitable drug polymer ratio for the study.

3. Development of nanoparticles based on studies in step 1 and 2.

Optimization of the formulations.

7.3. Does it require any investigation or interventions to be conducted or patients or other humans or animals? If so please describe briefly:

No.

7.4. Has ethical clearance been obtained from your institute in case of 7.3

Not applicable.

8.

List of references:

1. Davaran S, Rashidi MR, Pourabbas B, Dadashzadeh M and Haghshenas NM. Adriamycin release from poly(lactide-co-glycolide)-polyethylene glycol nanoparticles: synthesis and in vitro characterization. Int J Nanomedicine. 2006; 1(4):535–39.

2. Layre A-M, Couvreur P, Chacun H, Aymes-Chodur C, Ghermani N-E, Poupaert J, Richard J, Requier D and Gref R. Busulfan loading into poly(alkyl cyanoacrylate) nanoparticles: Physico-chemistry and molecular modeling. J Biomed Mater Res Part B: Appl Biomater. 2006; 79B (2):254-62.

3. Yan C-H, Yuan X-B, Kang C, Zhao Y, Liu J, Guo Y, Lu J, Pu P and Sheng J. Preparation of Carmustine-loaded PLA ultrasmall-nanoparticles by adjusting micellar behavior of surfactants. J Appl Polym Sci. 2008; 110 (4): 2446-52.

4. Sharma P, Ganta S, Denny WA and Garg S, Formulation and pharmacokinetics of lipid nanoparticles of a chemically sensitive nitrogen mustard derivative: Chlorambucil. Int J Pharm. 2009; 367(1-2):187-94.

5. Gryparis EC, Hatziapostolou M, Papadimitriou E and Avgoustakis K. Anticancer activity of cisplatin-loaded PLGA-mPEG nanoparticles on LNCaP prostate cancer cells. Eur J Pharm Biopharm. 2007; 67(1):1-8.

6. Janes KA, Fresneau MP, Marazuela A, Fabra A and Jose M, Chitosan nanoparticles as delivery systems for doxorubicin. J Control Release. 2001; 73(2-3):255-67.

7. Seo D-H, Jeong Y-II, Kim D-G, Jang M-J, Jang M-K and Nah J-W. Methotrexate-incorporated polymeric nanoparticles of methoxy poly(ethylene glycol)-grafted chitosan. Colloids Surf B Biointerfaces. 2009; 69(2):157-63.

8. Li F, Li J, Wen X, Zhou S, Tong Z, Su P, Li H and Shi D. Anti-tumor activity of paclitaxel-loaded chitosan nanoparticles: An in vitro study. Mater Sci Eng. 2009; 29(8):2392-97.

9. Zhenqing H, Heng W, Qian W, Qian S, Chunxiao Z, Chuanming Z, Xialong T and Qiqing Z. New Method to Prepare Mitomycin C Loaded PLA-Nanoparticles with High Drug Entrapment Efficiency. Nanoscale Res Lett. 2009; 4(7):732-37

10. Kwon H-Y, Lee J-Y, Choi S-W, Jang Y and Kim J-H. Preparation of PLGA nanoparticles containing estrogen by emulsification-diffusion method. Colloids Surf A Physicochem Eng Asp. 2001; 182(1-3):123-30.

11. Turos E, Reddy GSK, Greenhalgh K, Ramaraju P, Abeylath SC, Jang S, Dickey S and Lim DV. Penicillin-Bound Polyacrylate Nanoparticles: Restoring the Activity of (-Lactam Antibiotics Against MRSA. Bioorg Med Chem Lett. 2007; 17(12):3468-72.

12. Wang X, Chi N and Tang X. Preparation of estradiol chitosan nanoparticles for improving nasal absorption and brain targeting. Eur J Pharm Biopharm. 2008; 70:735-740.

13. Wu Y, Yang W, Wang C, Hu J and Fu S. Chitosan nanoparticles as a novel delivery system for ammonium glycyrrhizinate. Int J Pharm. 2005; 295:235-45.

9.

Signature of the candidate

(AMBIKA T. V.)

10.

Remarks of the guide:

Recommended for research and submission of dissertation.

11.

Name and Designation (in block letters)

11.1. Guide

11.2. Signature

dr. b. wilson,

Professor & head,

Department of Pharmaceutics,

Dayananda Sagar College of Pharmacy,

Kumaraswamy Layout,

Bangalore-560078.

11.3. Co-guide if any

Not applicable

11.4. Signature

11.5. Head of the department

11.6. Signature

dr. b. wilson,

Professor & Head,

Department of Pharmaceutics,

Dayananda Sagar College of Pharmacy,

Kumaraswamy Layout,

Bangalore-560078.

12.

12.1. Remarks of the principal

12.2 Signature

Dr. V. Murugan,

Principal,

Dayananda Sagar College of Pharmacy,

Kumaraswamy Layout,

Bangalore-560078